Embryo Microinjection: A Technique to Deliver a Compound into the Zebrafish Yolk
Published: April 30, 2023
Abstract
Source: Sorlien, E. L., et al. Efficient Production and Identification of CRISPR/Cas9-generated Gene Knockouts in the Model System Danio rerio. J. Vis. Exp. (2018).
This video describes a technique of Embryo microinjection to deliver compounds into Zebrafish yolk sac.
Protocol
1. Microinjection of CRISPR-components into Zebrafish Embryos Set up breeding tanks the night prior to injecting by placing the number of desired males and females (typically 2 females and 1 or 2 males) in a breeding tank with a divider in place. Prepare a microinjection plate with 1.5% agarose in 1x E3 media (see Table of Materials) with 0.01% methylene blue (a fungicide) by pouring 35 mL of the melted agarose into a 10 cm Petri dish and gently lay a plastic mold to create wedge-sh…
Representative Results
Figure 1: Comparison of the health of 24 hpf injected embryos. A living embryo (A) developed to 24 hpf, is easily distinguished from an embryo that has aborted development (B). Embryos that resemble (B) or have drastically altered features to (A), such as spinal curvature or altered head and eye development should be removed from dish
Materials
| RNase-free water | Any brand | Synthesis of guide RNAs. Thermo Fisher and Qiagen both carry suitable water. |
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| E3 embryo media | Made in house | Media for raising embryos and making injeciton plate. Recipe: http://cshprotocols.cshlp.org/ content/2011/10/pdb.rec66449 |
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| Methylene Blue | Sigma-Aldrich | M9140 | Added to 1x E3 media to prevent fungal growth on embryos. |
| Petri dish | Thermo Fisher | FB0875711Z | Store embryos, cast injection plate |
| Agarose | Denville | CA3510-8 | Casting injection plate, agarose gels. |
| Microinection mold | Adaptive Science Tools | TU-1 | To create wells to hold embryos during injection |
| Phenol Red | Sigma-Aldrich | P0290 | Dye for visualization of injection |
| Mineral Oil | Sigma-Aldrich | M5904-5ML | To calibrate needle injection volume. |
| Transfer pipettes | Any brand | Moving embryos | |
| Razor blade | Thermo Fisher | 11295-10 | Cutting injection needle, tail clipping adult fish. |
| Incubator | Maintaining embryos at 28.5 C. | ||
| Verticle pipette puller | David Kopf Instruments | 700C | Geneate needles for injection. Additional needle pulling instructions: http:// cshprotocols.cshlp.org/ content/2006/7/pdb.prot4651.long |
| Capillary tubes | Sutter Instruments | BF100-58-10 | Geneate needles for injection. |
| Microloader tips | Eppendorf | 930001007 | Load solution into injection needles |
| Microinjector | World Precision Instruments | PV 820 | Injecting embryos. |
| Disecting microscope | Leica | Injecting embryos. | |
| Microwave | Any brand | Casting injection plate, agarose gels. | |
| Scale | Any brand | Casting injection plate, agarose gels. | |
| Gloves | Any brand | For all aspects of the protocol. | |
| N2 | Any brand | To expell liquid from the capillary for embryo injection | |
| PCR strip tubes | Any brand | For all aspects of the protocol. | |
| 200 µL tips | Any brand | For all aspects of the protocol. | |
| 10 µL tips | Any brand | For all aspects of the protocol. | |
| Micropipettes | Any brand | For all aspects of the protocol. |
Diesen Artikel zitieren
Embryo Microinjection: A Technique to Deliver a Compound into the Zebrafish Yolk. J. Vis. Exp. (Pending Publication), e20196, doi: (2023).